Microfabricated reactor, process for manufacturing the reactor, and method of amplification

ABSTRACT

An integrated microfabricated instrument for manipulation, reaction and detection of microliter to picoliter samples. The instrument is suited for biochemical reactions, particularly DNA-based reactions such as the polymerase chain reaction, that require thermal cycling since the inherently small size of the instrument facilitates rapid cycle times. The integrated nature of the instrument provides accurate, contamination-free processing. The instrument may include reagent reservoirs, agitators and mixers, heaters, pumps, and optical or electromechanical sensors. Ultrasonic Lamb-wave devices may be used as sensors, pumps and agitators.

RELATED APPLICATIONS

This is a divisional of application Ser. No. 07/938,106 filed Aug. 31,1992 now U.S. Pat. No. 5,639,423. This application is a continuation ofour application Ser. No. 07/938,106 filed on Aug. 31, 1992, now U.S.Pat. No. 5,639,423. This application is also related to U.S. Pat. No.5,129,261, Ser. No. 07/467,412, issued Jul. 14, 1992, and applicationSer. No. 07/162,193, filed Feb. 29, 1988, now abandoned, for aPlate-mode Ultrasonic Sensor. The entire disclosures of theseapplications are hereby incorporated by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No.ECD-86-14900 (BSAC) awarded by the National Science Foundation. TheGovernment has certain rights to this invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to instruments for chemicalreaction control, product and reactant manipulations, detection ofparticipating reactants and resultant products, and more particularly tointegrated microfabricated instruments which perform microscale chemicalreactions involving precise control of parameters of the reactions. Theparameters of the reaction controlled by the instrument may betemperature, pressure, concentration of reactants, the intensity orfrequency of incident light, electromagnetic fields, or ultrasonicpressure waves, etc.

The term “integrated microfabrication” is used herein to refer to allprocesses used for batch production of semiconductor microelectronics,and all related microfabrication processes such as LIGA (see R. S.Muller, R. T. Howe, S. D. Senturia, R. L. Smith, and R. M. White, ed.MICROSENSORS, IEEE Press, 472 pages, 1990). Microfabricationtechnologies include, but are not limited to, sputtering,electrodeposition, low-pressure vapor deposition, photolithography andetching. Microfabricated devices are usually formed on crystallinesemiconductor substrates such as silicon or gallium arsenide.Noncrystalline materials such as glass or certain polymers may be usedalthough crystalline materials provide certain advantages. The shapes ofcrystalline devices can be precisely controlled since etched surfacesare generally crystal planes, and crystalline materials may be bonded byprocesses such as fusion at elevated temperatures or the field-assistedmethod (Mallory bonding). Materials which are not semiconductors, suchas quartz or glass, may be used, though semiconductor materials providethe advantage that electronic circuitry may be integrated into thesystem by the use of conventional integrated-circuit fabricationtechniques.

Monolithic microfabrication technology now allows the production ofelectrical, mechanical, electromechanical, optical, chemical and thermaldevices including pumps, valves, heaters, mixers and species detectorsfor microliter to nanoliter quantities of solids, liquids and gases.Microscale sensors include optical waveguide probes and ultrasonicflexural-wave sensors. The integration of these devices into a singlesystem allows for the batch production of microscale reactor-basedanalytical instruments. Integrated microinstruments may be applied tobiochemical, inorganic, or organic chemical reactions to performbiomedical and environmental diagnostics, and biotechnologicalprocessing and detection.

Such integrated microfabricated devices can be manufactured in batchquantities with high precision, yet low cost, thereby making recyclableand/or disposable single-use devices practical. Alternatively, theinstrument may consist of an array of reaction instruments which are tooperate in parallel to simultaneously perform a number of relatedreactions. Operation of such instruments is easily automated, furtherreducing costs. Since the analysis can be performed in situ, thelikelihood of contamination is very low. Because of the inherently smallsizes of such devices, the heating and cooling can be extremely rapid,and the devices can have very low power requirements. Such devices maybe powered by batteries or by electromagnetic, capacitive, inductive oroptical coupling.

Small volumes and high surface-area to volume ratios providemicrofabricated reaction instruments with a high level of control of theparameters of a reaction. Heaters may produce temperature cycling orramping, sonochemical and sonophysical changes in conformationalstructures may be produced by ultrasound transducers, andpolymerizations may be generated by incident optical radiation.

Synthesis reactions, and especially synthesis chain reactions, such asthe polymerase chain reaction (PCR) or the ligase chain reaction, areparticularly well-suited for microfabricated reaction instruments. PCRcan selectively amplify a single molecule of DNA (or RNA) of an organismby a factor of 10⁶ to 10⁹. This well-established procedure requires therepetition of heating (denaturing) and cooling (annealing) cycles in thepresence of an original DNA target molecule, specific DNA primers,deoxynucleotide triphosphates, and DNA polymerase enzymes and cofactors.Each cycle produces a doubling of the target DNA sequence, leading to anexponential accumulation of the target sequence. PCR-based technologyhas been applied to a variety of analyses, including environmental andindustrial contaminant identification, medical and forensic diagnostics,and biological research.

The procedure involves: (1) processing of the sample to release targetDNA molecules into a crude extract; (2) addition of an aqueous solutioncontaining enzymes, buffers, deoxyribonucleotide triphosphates (dNTPS),and oligonucleotide primers; (3) thermal cycling of the reaction mixturebetween two or three temperatures (e.g., 90-96, 72, and 37-55° C.); and(4) detection of amplified DNA. Intermediate steps which incorporatesignal-producing and/or surface-binding primers, or which purify thereaction products, via, for example, electrophoresis or chromatographymay be introduced. A problem with standard PCR laboratory techniques isthat the PCR reactions may be contaminated or inhibited by introductionof a single contaminant molecule of extraneous DNA, such as those fromprevious experiments, or other contaminants, during transfers ofreagents from one vessel to another.

PCR reaction volumes are presently typically on the order of 50microliters. A thermal cycle typically consists of heating a sample to afirst temperature, maintaining the sample at the first temperature,cooling the sample to a second lower temperature, and maintaining thetemperature at that lower temperature. The rate at which the sample isheated is generally limited by the heater rather than the rate of heattransfer to the sample. Presently, each of the four stages of a thermalcycle requires approximately one minute, and the time required fortwenty to forty complete thermal cycles is therefore from about one tothree hours. The cycling time has been reduced by performing the PCRreaction in capillary tubes (see C. T. Wittwer, G. C. Fillmore, and D.J. Garling, Analytical Biochemistry, 186, pp. 328-331 (1990)). Ahigh-power forced air heater was used to heat the tubes. The thinnestcapillary tubes contained a sample volume of about ten microliters. Eachcycle consisted of a heating step, a waiting period, a cooling step andanother waiting period, and each step required approximately fifteenseconds.

Although the PCR reaction requires thermal cycling of the reagents, anyreaction that benefits from precise temperature control, and/or rapidthermal cycling, thermal ramping, or any other temperature variation ofreagents with time (hereinafter to be referred to as temperatureprogramming) will be well suited for the microfabricated reactioninstrument of the present invention.

An object of the present invention is therefore to provide a integratedmicrofabricated reactor.

Another object of the present invention is to provide a reactor-basedinstrument for inorganic, organic and biochemical reactions, and inparticular for diagnostics.

Another object of the present invention is to provide a reactor whichprovides high-precision control of reaction parameters.

Another object of the present invention is to provide a reactor whichprovides high-precision temperature control.

Another object of the present invention is to provide a reactor whichprovides rapid high-precision thermal cycling, ramping or programming.

Another object of the present invention is to provide a closed systemreactor which is self-contained, e.g. which is shipped from the factorycontaining the reagents, thereby minimizing the susceptibility tocontamination.

Another object of the present invention is to provide low-cost reactionand/or detection systems.

Another object of the present invention is to provide an instrument forin situ reactions which may be powered by incident electromagneticradiation or batteries.

Another object of the present invention is to provide arrays ofmicrofabricated reaction chambers which may operate in parallel orseries.

Additional objects and advantages of the invention will be set forth inthe description which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and obtained by means ofthe instrumentalities and combinations particularly pointed out in theclaims.

SUMMARY OF THE INVENTION

The present invention is directed to an instrument for in situ chemicalreactions in a microfabricated environment. The instrument is especiallyadvantageous for biochemical reactions which require high-precisionthermal cycling, particularly DNA-based manipulations such as PCR, sincethe small dimensions typical of microinstrumentation promote rapidcycling times.

The present invention provides a reaction instrument comprised ofintegrated microfabricated elements including a reagent chamber and ameans for manipulating the reaction of the reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, schematically illustrate a preferredembodiment of the invention and, together with the general descriptionof the preferred embodiment given below, serve to explain the principlesof the invention.

FIG. 1 shows a cut-away perspective view of a reaction instrument of thepresent invention mounted in a power source/control apparatus.

FIG. 2 is a schematic of a reaction instrument of the present invention.

FIG. 3 shows a cross-sectional view of a reaction chamber of the presentinvention.

FIGS. 4( a) through 4(f) show cross-sectional views of the stages offabrication of a reaction chamber of the present invention.

FIG. 5 shows a top view of a reaction chamber (dashed outline) below thepiezoelectric and ground plane layers.

FIG. 6 a shows the typical flow velocity profile field for a fluidforced through a conduit with static bottom and side surfaces, and FIG.6 b shows the flow velocity profile for a fluid pumped through a conduitby a flexural-wave pump.

FIG. 7 shows gel electrophoretic results verifying the amplification ofan HIV genetic sequence in a microfabricated reaction chamber of thepresent invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The microinstrument of the present invention has integratedmicrofabricated components that perform reactant and productmanipulations and detection on microliter to picoliter samples. Samplesmay be less than a milliliter, less than a microliter, or less than apicoliter. By the selection and integration of appropriatemicrofabricated devices, a precise and reliable reaction and analysisinstrument for PCR-based diagnostics is implemented.

The instrument may be fabricated in a wide variety of different forms.Many microinstruments may be manufactured on a single wafer and can runin parallel, allowing the processing and analysis of several targetagents and controls simultaneously. Individual small and disposabledies, each a complete microinstrument, may be fabricated. The device maybe fabricated to allow a continual flow of reagents through theinstrument. A reagent reservoir of the microinstrument may have a thinsilicone rubber wall so that the reagent may be inserted into themicroinstrument by a hypodermic needle.

Alternatively, a needle may be integrated into the microinstrument sothat a patient can be pricked by the needle and a blood sample, or anyother type of body fluid, will directly enter the instrument. Anintegrated needle can also be used to extract body fluids from plantsand animals. The reagent may also be loaded into the microinstrument bypipetting, a modified ink-jet printing process, or other means at thefactory. The reagent may be lyophilized or dried, or previously storedin the chamber.

Detection signals may be processed and stored by integratedmicroelectronic devices so that result interpretation and controlmechanisms (which may utilize feedback) can be integrally contained onthe microinstrument. The low power needs of microinstrumentation allowssuch systems to be powered by incident electromagnetic radiation, lowvoltage batteries, or incident optical radiation converted to electricalenergy by on-board photocells.

The components of a microinstrument may include reservoirs for retainingreagents, agitators and mixers, heaters to perform denaturing andannealing cycles, pumps, optical and/or electromechanical sensors todiscriminate reagents, and reagent separators. Microheaters may beresistive heaters consisting of materials such as polysilicon patternedonto and made an integral part of the microstructure. The micropumps maybe Lamb-wave devices (see U.S. Pat. No. 5,006,749, R. M. White, 1991),electrokinetic pumps, or other microfabricated pump structures. Themicrodetection instruments may be fluorescence-based optical fiberspectroscopes which utilize microfabricated light sources and detectors(e.g., LEDs or diode lasers and detectors); Lamb-wave sensors (see U.S.Pat. No. 5,129,261, Ser. No. 07/467,412 filed Jan. 16, 1990, andapplication Ser. No. 07/775,631 filed Oct. 10, 1991); electrochemicaldetection of biochemical molecules by surface plasmon resonance or otherprocesses involving immobilized biochemicals; electrochemical sensingdevices; or other appropriate detection methodologies. Surfacetreatments may be applied to components of the device for reactionenhancement, product separation, and species detection. The surfacetreatments can be based on numerous well-known procedures such assilanol-based derivatizations or other appropriate treatments. Chemicalspecies separators can utilize microelectrophoresis either in acapillary or within a gel, or can be based on other appropriatemethodologies.

One embodiment of the present invention performs the polymerase chainreaction (PCR). The minute reagent volumes and the specific reactionsequence of the PCR technique play favorably into the advantages of thepresent invention. The integrated microsystem provides a highlyautomated, miniaturized, analytical instrument for very rapid in situanalyses and production of a variety of samples. Integratedmicrofabricated PCR instruments are capable of performing, in situ, manyreactions and manipulations with precise control of temperature,evaporation, small-volume reagent delivery, product separation,isolation and detection. Such highly automated technologies shouldgreatly expedite the use of DNA-based technologies for biomedical (e.g.,the Human Genome Project); environmental (e.g., contaminantidentification); industrial (e.g., biotechnology); and forensicapplications. The principles applied and problems solved in developing aPCR instrument may of course be applied to other chemical reactions,analyses, and syntheses, especially other biochemical reactions,analyses, and syntheses.

PCR in a microdevice can be just one step in a series of manipulationsleading to the diagnostic detection of a variety of target species orthe use of PCR products in genetic engineering. Physical and chemicaltreatments such as those described below can also be incorporated intothe pre-PCR and post-PCR phases of microdevice-based treatments toaugment the reactions in situ. Amplification via PCR yields productsthat may be subject to further enhancement or utilized for detection ofother chemicals. Physical and chemical control via microdevices ofbiological cells and reagents prior to and after the production of PCRproducts will expand the number of the potential applications ofDNA-based processes and analyses.

Pre-PCR manipulation of target cells or microorganisms can beaccomplished with microdevices of the present invention. For exampleultrasonic waves may be applied to disrupt and expose cell componentsthrough lysis, and to unravel large or long chain molecules such as DNAand proteins via disruption of secondary structure. Cell lysis may alsobe induced electrically or chemically. Ultrasonic waves and surfacechemistry treatments can be used to manipulate cells and cell-contents,as can chemical treatment by stirring and/or mixing reagents from otherchambers on the microinstrument. Sonication on a macro-scale inconjunction with agitating microparticles, for example, has been used tofacilitate the extraction of DNA from parafin-embedded fixed cells (M.J. Heller, L. J. Burgart, C. J. Ten Eyck, M. E. Anderson, T. C. Greiner,and R. A. Robinson, Biotechniques, 11, #3, 1991, pp. 372-377).Strategies similar to this which rely on the inherent properties of amicrodevice can be used to process intact cells, microorganisms,tissues, and other analytical samples for PCR and subsequent techniques.

Potential post-PCR treatments by the microdevice are also numerous. Itshould be noted that PCR is often an integral part of the potentialapplication of a device to further biotechnological manipulations andanalyses. Once PCR has been performed, post-PCR manipulations can leadto a myriad of possible microdevice-based DNA analyses and treatments. Afew examples of such analyses are: large-scale and small-scale DNAsequencing of target species, cell-typing, analysis of PCR products withDNA probes, DNA recombination, DNA fingerprinting, DNA cloning, cellcloning, physical mapping of genes, incorporation of genetic vectors,genetic therapy, treatment and testing of biotechnological processes,and the maintenance of DNA libraries. Such analyses can lead to the useof DNA as vectors to produce cells or other biological entities to makedesired products such as proteins or other compounds, or it can be usedto produce DNA for use in therapies or biotechnological processes.

PCR products may also be manipulated in order to be incorporated intogenetic engineering vectors such as plasmids. The vectors maysubsequently be incorporated into target cells for the production ofdesired compounds. The target cells or moieties and reagents can bestored in reservoirs on the device and released for exposure to thevectors when the proper physical/chemical conditions have beenestablished. One other potential application would be the in situ (invitro or in vivo) release of PCR products for direct genetic therapy ormanipulations. Direct DNA sequencing of PCR products (single ordouble-stranded) can be accomplished with the use of unique temperature,enzymatic, or other separation schemes and detection methodologies; allof which can be incorporated into a microdevice.

Detection windows, reflective and absorptive surfaces, optic sources andother optical components can be fabricated and integrated onto amicrodevice instrument, providing optical detection capabilities. Thestatus of a reaction may be monitored by illuminating the reagentsthrough an optical window and measuring absorption and/or luminescence.A waveguide may be fabricated by depositing a plane of transparentmaterial between semiconducting planes, or by bonding two waferstogether, at least one of the wafers having a transparent surface layer.The optical path of the waveguide is parallel to the fabricationsurface. Alternatively, a window with an optical path parallel to thenormal vector of the fabrication surface may be constructed usingstandard patterning techniques. Data analyses can be accomplished withon-board electronics which may provide electronic or optical output.

Analysis of PCR products, sequences of target DNA, or syntheticanalogues in microdevices can be accomplished with the manipulativecapabilities of microfabricated electrical and mechanical machines. Forexample, two-dimensional arrays of predetermined DNA sequences (probes)can be used to detect or verify PCR products, and their subsequentanalyses can be accomplished with microdevices.

As shown in FIG. 1, an embodiment 20 of the present invention is shownabove a recess 105 in a power source/control system 100. A hypodermicneedle 110 is shown inserting a sample through a silicone rubber window120 into a reaction instrument 20. The reaction is controlled andpowered by: inductive coupling, such as that between coil L_(CL) in theinstrument 20 and a magnetic coil 130 as shown in FIG. 1; by capacitivecoupling, such as that between the plates of capacitor C₃ and plates 140a and 140 b; and by electromagnetic coupling between resonant circuit(not shown) in the instrument 20 and a radio-frequency antenna 135.

A schematic of a preferred embodiment 20 of the present invention isshown in FIG. 2. Three reagent chambers 10, 12 and 14 contain reactants.One chamber 10 contains the DNA primers, one chamber 12 contains thepolymerase, and one chamber 14 contains the nucleotides and anydetection-tag molecules, such as magnetic beads. The contents of thechambers 10, 12 and 14 have been loaded at the factory. The target DNAmolecule is placed in reagent chamber 10 by insertion of a hypodermicneedle or the like through silicone rubber window 120. The window 120may alternatively be composed of any other type of appropriate naturalor synthetic elastomer. The reactants in the reagent chambers 10, 12 and14 are connected by channels 22, 24 and 26 to a reaction chamber 30.Typically the chambers 10, 12, 14 and 30 have a volume ranging frommicroliters to nanoliters. The channels 22, 24 and 26 are equipped withLamb-wave pumps LW₁, LW₂ and LW₃, respectively, for pumping thereactants in the reactant chambers 10, 12 and 14 in the directions ofthe arrows to the reaction chamber 30. The Lamb-wave pumps may belocated on any wall, or on multiple walls, of the channels 22, 24 and26. Lamb-wave pump LW₁ is connected to a capacitor C₁. Similarly theother two Lamb-wave pumps LW₂ and LW₃ are connected to capacitors C₂ andC₃, respectively.

The surface tension across the narrow midsections of the channels 22, 24and 26 prevents the reactants from flowing into the reaction chamber 30until pumping is initiated. The surfaces of the channels 22, 24 and 26may be treated to raise the surface tension thereby further inhibitingflow of reagents when the pumps LW₁, LW₂ and LW₃ are not activated.

The reaction chamber 30 may be equipped with a Lamb-wave transducerLW_(C) and a heater H_(C). The Lamb-wave transducer is connected toinductor L_(CL). The heater H_(C) is connected to a resonant circuitconsisting of an inductor L_(CH) and a capacitor C_(CH). The Lamb-wavetransducer LW_(C) acts as an agitator, mixer, or sonochemical inducer.

A channel 32 connects the reaction chamber 30 to a detection chamber 34.The channel 32 is equipped with a Lamb-wave pump LW_(DP), which isconnected to a resonant circuit consisting of an inductor L_(DP) and acapacitor C_(DP). The detection chamber 34 is equipped with a Lamb-wavesensor LW_(D). The Lamb-wave sensor LW_(D) is connected to a capacitorC_(D).

For ease of notation, an exemplary Lamb-wave device chosen from the setLW₁, LW₂, LW₃, LW_(C), LW_(DP), and LW_(D) will be denoted by LW and thecorresponding capacitor and/or inductor electrically connected to theLamb-wave device will be denoted by C and L, respectively, hereinafter.Lamb-wave transducers have high mechanical Q values and can therefore bepowered by only a narrow range of alternating voltage frequencies. TheLamb-wave pumps LW₁, LW₂, and LW₃, and the Lamb-wave sensor LW_(D) arepowered capacitively by generating an electric field between the plates140 a and 140 b at the resonant frequencies of the Lamb-wave transducersLW₁, LW₂, LW₃, and LW_(D). The alternating frequency electric fieldsgenerate alternating frequency voltages across the capacitors C₁, C₂, C₃and C_(D), and Lamb waves at this frequency in the transducers LW₁, LW₂,LW₃ and LW_(D). But because the transducers LW₁, LW₂, LW₃, and LW_(D)have high Q values, only when the frequency of the imposed field is nearthe resonant frequency of a transducer does the transducer vibrate withany substantial magnitude. Similarly, the Lamb-wave mixing chambertransducer LW_(C) is powered by an alternating frequency magnetic fieldgenerated by the coil 130 at the mechanical resonant frequency of thetransducer LW_(C). The heater H_(C) and the Lamb-wave pump LW_(DP) areactivated by directing an electromagnetic wave from the antenna 135 tothe resonant circuits C_(CH) and L_(CH), and C_(DP) and L_(DP),respectively. The frequency of the incident electromagnetic radiationmust correspond to the resonant frequency of the electrical elementsC_(DP), L_(DP) and LW_(DP), and must also correspond to the mechanicalresonant frequency of the transducer LW_(DP), to activate the pumpLW_(DP). The frequency of the incident electromagnetic radiation mustcorrespond to the resonant frequency of the electrical elements C_(H),L_(CH) and H_(C) to activate the heater H_(C).

The PCR reaction is initiated by pumping the reagents in the reagentchambers 10, 12 and 14 along the directions of the arrows to thereaction chamber 30 by activating the reagent pumps LW₁, LW₂ and LW₃. Aseries of approximately twenty to forty thermal cycles are theninitiated, during each cycle the temperature of the reactants in thereaction chamber 30 goes from 55° C. to 96° C., and back to 55° C., forexample. The temperature of the reaction chamber 30 is determined by thepower of the incident electromagnetic signal at the frequencycorresponding to the resonant frequency of the circuit comprised of thecapacitor C_(CH), and the inductor L_(CH), together with the heaterH_(C). The reaction chamber 30 Lamb-wave device LW_(C) acts as anagitator or mixer as described below to mix the reagents and promote thereaction.

When the thermal cycling is complete the contents of the reactionchamber 30 are pumped by Lamb-wave pump LW_(DP) in the direction of thearrow to the detection chamber 34. The preferred embodiment utilizes aLamb-wave sensor LW_(D). Alternatively, the detection chamber may beprovided with an optical window and testing may be performed byfluorescence-based or absorption-based optical spectroscopy.

A cross-sectional view taken along line 3-3 of FIG. 2 of the reactionchamber 30 is shown in FIG. 3. FIG. 4( a)-(f) show cross-sectional viewsof the bottom half of the chamber during successive stages of formationof the chamber 30 from a silicon substrate 50 b. A similar set of stagesare involved in the fabrication of the top portion of the chamber 30.Once fabricated, the top and bottom portions may be bonded together byMallory bonding.

The chamber cavity 31 is bounded by a ceiling 70 t, a floor 70 b, andside walls consisting of silicon sections 50 t and 50 b and siliconnitride sections 49 tl, 49 bl, 49 tr and 49 br. The height of thechamber cavity 31 is approximately 0.5 mm, and the width and length ofthe chamber are approximately 4 mm or less. The whole instrument 20 mayfit on a wafer as small as 1 cm×1 cm×0.5 cm.

The indentations in the silicon substrates 50 b and 50 t are formed bycoating one side of the substrates 50 b and 50 t with silicon nitridelayers 52 b and 52 t, and patterning silicon nitride layers on the othersides of the substrates 50 b and 50 t to form sections 49 bl, 49 br, 49tl, and 49 tr, respectively, as shown in FIGS. 3 and 4( a). Thesilicon-nitride layers 52 t and 52 b are preferably applied bylow-pressure chemical-vapor deposition of the silicon-nitride, and arepreferably a low-stress nitride. The thickness of silicon-nitride layers52 t and 52 b is chosen to provide a balance between the mechanicalstrength of the layers which increases with thickness, and thesensitivity of the Lamb-wave detector which decreases with thickness.The thickness is also chosen to provide practical resonant frequenciesfor the device. The thickness of the silicon-nitride layers 52 t and 52b is preferably about 3 μm, plus or minus ten percent.

The system is etched, for example with the wet chemical etchant KOH, tocreate a cavity in the silicon substrate 50 as shown in FIG. 4( b). Theremaining sections of silicon 50 b form portions of the side walls ofthe cavity 31.

Located outside the reaction chamber cavity 31 to the exterior ofsilicon nitride layers 52 b and 52 t are top and bottom polycrystallinesilicon layers 54 t and 54 b, respectively. The polycrystalline layers54 t and 54 b are deposited on the silicon nitride layers 52 b and 52 tby chemical vapor deposition and selectively patterned. The bottomportion of the chamber 30 with the patterned polycrystalline layer 54 bon the silicon nitride layer 52 b is shown in FIG. 4( c). The thicknessof the polycrystalline layers 54 t and 54 b is preferably between 2000and 4000 angstroms, and more preferably 3000 angstroms, plus or minusfive percent.

Top and bottom barrier layers, composed of an insulating material suchas low-stress silicon nitride or silicon dioxide, are deposited bylow-temperature oxidation and patterned. The lower barrier layers 58 bl,58 bcl, 58 bc, 58 bcr and 58 br lie to the exterior of thepolycrystalline layer 54 b and the silicon nitride layer 52 b as shownin FIG. 4( d). Similarly, the upper barrier layers 58 tl, 58 tcl, 58 tc,58 tcr, and 58 tr lie to the exterior of the polycrystalline layer 54 tand the silicon nitride layer 52 t. Left and right bottom access holes64 bl, 64 br, 65 bl, and 65 br provide access to the bottompolycrystalline layer 54 b (and similarly for the top access holes 64tl, 64 tr, 65 tl, and 65 tr, (not shown)). The thickness of the barrierlayers 58 cl, 58 tcl, 58 tc, 58 tcr, and 58 tr, (to be collectivelyreferred to as 58 t) and 58 bl, 58 bcl, 58 bc, 58 bcr and 58 br (to becollectively referred to as 58 b) is preferably between 1000 and 5000angstroms, and more preferably 2500 angstroms, plus or minus tenpercent. The silicon nitride layers 52 b and 52 t and the barrier layers58 b and 58 t thermally isolate the polycrystalline layers 54 b and 54 tfrom the high conductivity silicon layers 50 b and 50 t, therebyincreasing the effectiveness of the heaters formed by thepolycrystalline layers 54 b and 54 t.

As shown in FIGS. 3 and 4( e), top left and right conducting leads 56 tland 56 tr, bottom left and right conducting leads 56 bl and 56 br, topleft and right transducers 60 bl and 60 tr, bottom left and righttransducers 60 bl and 60 br, top left and right four-contact resistancemonitoring leads 67 tl and 67 tr, and bottom left and right four-contactresistance monitoring leads 67 bl and 67 br are then patterned onto thedevice. Through access holes 64 tl and 64 tr, the leads 56 tl and 56 trmake electrical contact with the polycrystalline layer 54 t, andsimilarly for the bottom section of the chamber 30. Through access holes65 tl and 65 tr (labelled in FIG. 4( d) but not in FIG. 4( e)), theleads 67 tl and 67 tr make electrical contact with the polycrystallinelayer 54 t, and similarly for the bottom section of the chamber 30. Thetop polycrystalline layer 54 t is therefore electrically connected toleads 56 tl, 56 tr, 67 tl and 67 tr. Similarly, the bottompolycrystalline layer 54 b is electrically connected to lead 56 bl, 56br, 67 bl and 67 br. Current passing through polycrystalline layers 54 tand 54 b generates heat. Therefore, the temperature of the chamber 30can be controlled by the amount of voltage applied across the top andbottom leads 56 tl and 56 tr, and 56 bl and 56 br, respectively. Becausethe exterior temperature is generally below that of the chemicalreactions the system is cooled by the ambient air and cooling elementsare not generally required.

The temperature of the system is monitored by measurement of theresistance of the polycrystalline layers 54 t and 54 b by connectingleads 67 tl and 67 tr, and 67 bl and 67 br of four-contact resistancemeasuring circuits (not shown) to the top and bottom polycrystallinelayers 54 t and 54 b, respectively. The resistance increases linearlywith temperature.

FIG. 5 depicts a view of the bottom of the chamber 30 subsequent to thedeposition of the transducers 60 bl and 60 br, leads 56 bl and 56 br,and four-contact resistance leads 67 bl and 67 br, as shown in FIG. 4(e). For clarity not all transducer leads 64 blx, 64 bly, 64 brx and 64bry shown in FIG. 5 are depicted in FIGS. 3, 4(e) and 4(f). Only sixfingers per transducer 60 bl and 60 br are shown in FIG. 5 for clarity,though transducers having approximately twenty or more fingers arepreferred. The cavity 31 and channels 24 and 32 lay beneath the barrierlayer 58 b in the region between the dashed lines 29 and the crossbarsof the T-shaped leads 56 bl and 56 br. The access holes 64 bl and 64 br(not shown in FIG. 5) lie beneath the cross-bars of the leads 56 bl and56 br, and the access holes 65 bl and 65 br (not shown in FIG. 5) liebeneath the cross-bars of the leads 67 bl and 67 br, respectively. Thebottom left Lamb-wave transducer 60 bl consists of a plurality ofinterlaced fingers which are electrically connected to a pair oftransducer leads 64 blx and 64 bly. The top left and right transducers60 tl and 60 tr and the bottom left and right transducers 60 bl and 60br have similar shapes. The bottom transducers 60 bl and 60 br and thebottom transducer leads 64 bly, 64 blx, 64 bry and 64 brx, and the leads56 bl, 56 br, 67 bl and 67 br are aluminum and have a thickness ofpreferably 2000 to 6000 angstroms, and more preferably a thickness of4000 angstroms, plus or minus ten percent. The bottom transducers 60 bland 60 br and leads 56 bl, 56 br, 67 bl, and 67 br may be alternativelybe made of low-temperature oxide, or any other appropriate conductingmaterial. The top transducers 60 tl and 60 tr and leads 56 tl, 56 tr, 67tl, and 67 tr may be similarly constructed.

As shown in FIGS. 3 and 4( f), a bottom piezoelectric layer 62 bextending between and covering leads 67 bl and 67 br, fingers of thebottom transducers 60 bl and 60 br, and portions of leads 56 bl and 56br is then deposited and patterned. Similarly, a top piezoelectric layer62 t extending between and covering leads 67 tl and 67 tr, fingers ofthe bottom transducers 60 tl and 60 tr, and portions of leads 56 tl and56 tr is then deposited and patterned. The piezoelectric material may beZnO or any other appropriate material. The thickness of thepiezoelectric sections 62 t and 62 b is preferably between 0.5 and 2.0μm, more preferably 1 μm, plus or minus ten percent.

The piezoelectric sections 62 t and 62 b are covered by top and bottomconducting ground planes 66 t and 66 b, respectively, as shown in FIGS.3 and 4( f). The ground planes 66 t and 66 b may be aluminum, or anyother appropriate conducting material. The thickness of the conductingground plane layers 66 t and 66 b is preferably between 2000 and 6000angstroms, and more preferably 4000 angstroms, plus or minus tenpercent.

Lamb waves, also known as plate-mode waves, have a compressionalcomponent and a shear component. Together the components of a travelingLamb-wave in a slab can transport liquids and gases adjacent the slab,much the same way ocean waves transport a surfing board. Lamb-wavedevices can therefore act as pumps. Material near the surface of theslab has the maximum velocity since there are essentially no boundarylayer effects. This is extremely advantageous for the small geometriesassociated with microdevices. FIG. 6 a shows the typical flow velocityof a fluid through a conduit with two planar walls to illustrate theeffect of the friction induced by walls on the flow. The velocityprofile is parabolic, and at the left and right edges of the conduit thevelocity drops to zero due to friction between the fluid and the walls.The friction between the walls and the fluid reduces the efficiency ofthe pumping. In contrast, FIG. 6 b shows the flow velocity of a fluidthrough the same conduit when the front and back walls are Lamb-wavepumps. The flow velocity is almost constant between the right and leftwalls. The viscosity of the fluid transmits the momentum of the fluidnear the Lamb-wave pumps to the fluid farther from the pumps.

Lamb waves require a propagation medium which is at most severalwavelengths thick. The Lamb waves in this invention have frequencies inthe approximate range of 1 to 200 MHz, and typical pumping velocitiesfor a Lamb-wave device operated with a 10 volt transducer voltage are300 μm/sec for water, and 2 cm/sec for air.

The layers 54 t, 58 t, 62 t and 66 t which border the top of the chambercavity 31 will hereinafter be referred to as the ceiling 70 t of thechamber 30, and the layers 54 b, 58 b, 62 b and 66 b which border thebottom of the chamber cavity 31 will hereinafter be referred to as thefloor 70 b of the chamber 30. Lamb waves are generated in the ceiling 70t of the chamber 30 by applying an alternating voltage between atransducer lead, for instance the upper left 64 bly of FIG. 5, and thebottom ground plane 66 b. The transducer electrodes 60 bl differentiallydeform the piezoelectric material 62 b to produce a mechanical wavemotion in the ceiling 70 b. The amplitude of the Lamb waves is increasedby applying a second alternating voltage which is 180° out of phase tothe transducer lead 64 blx connected to the set of interlaced fingers.

Traveling waves are generated in the Lamb-wave pumps LW₁, LW₂, LW₃, andLW_(DP) in the directions of the arrows of FIG. 2 by application ofalternating voltages to the pumps LW₁, LW₂, LW₃, and LW_(DP) at thearrow-tail side. Standing waves are generated in the Lamb-wave detectorLW_(D) by application of in-phase alternating voltages electrodes atboth sides of the chamber. By sending Lamb-waves from left to rightacross the ceiling 70 t of the mixing chamber 30 (by application of analternating voltage between top left transducer 60 tl and the top groundplate 66 t) and Lamb waves from right to left across the floor 70 b ofthe chamber 30 (by application of an alternating voltage between bottomright transducer 60 br and the bottom ground plate 66 b), a stirring orcirculating action can be produced.

The phenomenon responsible for the operation of the Lamb-wave sensorLW_(D) in the detection chamber 34 is elastic wave propagation along amedium whose characteristics can be altered by a measurand, such asviscosity or density of the ambient fluid or gas. Where thecharacteristics of the waves propagating along the medium are dependentupon the characteristics of the propagation medium, the wavecharacteristics can be monitored or measured to provide an indication ofthe measurand value. For example, when the device absorbs vapors orgases from the atmosphere in a film deposited on the surface, the outputfrequency changes. Tests and analysis indicate that Lamb-wave sensorsare at least an order of magnitude more sensitive than other types ofacoustical vapor sensors operating at the same wavelength. The type ofDNA in an ambient fluid can be determined by measuring the viscosity asa function of temperature, since the denaturing temperatures ofdifferent types of DNA are well known.

Since Lamb-wave sensors can operate while in contact with a liquid suchas water, their use as biosensors is very significant. For instance, thesurface of a Lamb-wave sensor may be coated with single strands of aparticular DNA molecule. If the device is immersed in a solutioncontaining that same type of DNA molecule, the molecules in solutionwill pair with the molecules on the surface, increasing the mass of themembrane and therefore decreasing the frequency of oscillation. Also,the membrane may be made of a porous and permeable material, allowingthe coating of a greater surface area and also allowing the liquid to beflowed through the membrane, in order to speed up the DNA attachment.Intercalating dyes, such as ethidium bromide, may be used to augmentviscosity changes which occur during a reaction, thereby increasing thesensitivity of the sensor. Other biological interactions may also besensed.

FIG. 7 shows gel electrophoresis results verifying the amplification ofa specific HIV nucleic acid sequence performed by the microfabricatedreaction instrument of the present invention. The two outside bands C₁and C₂ represent calibrating size standards, and the three bandslabelled as D₁ represent the DNA amplified in the microreactor. The twobands E₁ to the left of center are results obtained with a commercialPCR thermocycler instrument from the same reaction mixture as that inthe microreactor. Since bands E₁ and D₁ are at the same height it isindicated that the microreactor has produced the correct target. Thethermal cycles of the commercial instrument were 4 minutes long. Thoseof the microreactor of the present invention were 1 minute in length.

In summary, an apparatus and method for in situ chemical reactions in amicrofabricated instrument has been described. It has been shown thatthe instrument facilitates extremely rapid thermal cycling andhigh-precision temperature control of microliter to nanoliter volumes.The apparatus and method are well suited for DNA-based reactions, suchas PCR. It has also been shown that such integrated devices minimize thepossibility for contamination of the reactions and may be operated at adistance by electromagnetic fields.

The present invention has been described in terms of a preferredembodiment. However, the invention is not limited to the embodimentdepicted and described. Many variations within the scope of the presentinvention are possible. For instance, the instrument may consist of aseries of chambers at different temperatures, and the temperatureprogramming of the reagents may be accomplished by transport of thereagents through the series of chambers. The instrument may have aninput channel and an output channel, and may be adapted to provide acontinual flow-through synthesis. Thermal cycling may be accomplished byrepeated transfer of reagents between two or more chambers which areheated to different temperatures.

In another alternate embodiment the monitoring of the temperature of achamber is accomplished at a distance by connecting an LC circuit to aprobe across a polycrystalline heating layer and measuring the Q-factorof the circuit. The Q-factor is measured by exciting the circuit with anincident electromagnetic field at the resonant frequency of the circuitand monitoring the time decay of the resonance, or by measuring thebandwidth of the circuit by applying a frequency modulated incident EMfield and measuring the circuit's frequency response. Since the measuredQ-factor is inversely proportional to the resistance and the resistanceincreases linearly with temperature, the temperature may be determinedby the measured Q-factor.

Other possible variations within the spirit of the invention include:the dimensions of components of the instrument are not limited to thosedisclosed; more or fewer reactants may be used and the reactants may beorganic such as a protein inorganic, or a combination of organic andinorganic; the reactants may be any type of large molecules, proteins,polymers or biochemical compounds; the detection means may be located inthe same chamber as the mixing means; the number of thermal cycles maybe greater than ten, or greater than twenty five; components of thedevice may be made of semiconducting materials other than silicon, or ofquartz, glass, polymers or other materials; the microfabricatedinstrument may be formed by the bonding of two wafers; the instrumentmay be provided with optical windows for optical monitoring of thereaction; the instrument may be controlled by direct electrical couplingof control circuitry to the leads of the pumps, heater and sensor; asilicone-rubber window may form a penetrable wall of any chamber orchannel; the instrument may be fabricated from a silicon-based material;the instrument may be powered by an integrated microfabricated battery;any Lamb-wave transducer may be activated by capacitive, inductive,electromagnetic or optical means; or electrokinetic pumps, or any otherappropriate type of pumping means, may be substituted for the Lamb-wavepumps.

Accordingly, the scope of the invention is defined by the appendedclaims.

1. A device for amplifying a preselected polynucleotide in a sample byconducting a polynucleotide amplification reaction, the devicecomprising: a solid substrate which is microfabricated to define: asample inlet port; a flow system for micro- to picoliter volumes,comprising: a sample flow channel extending from said inlet port; and apolynucleotide amplification reaction chamber in fluid communicationwith said flow channel; said chamber and said channel being ofdissimilar dimension; and a fluid exit port in fluid communication withsaid flow system; and means for thermally cycling the contents of saidchamber whereby in each cycle the temperature is controlled todehybridize double stranded polynucleotide and to permit the in-vitroamplification of a preselected polynucleotide.
 2. The device of claim 1,wherein said amplification reaction is a polymerase chain reaction(PCR), and wherein said amplification chamber contains: a preselectedpolynucleotide, a polymerase, nucleoside triphosphates, a first primerhybridizable with single strand of said polynucleotide, and secondprimer hybridizable with a nucleic acid comprising a sequencecomplementary to said single strand, wherein said first and secondprimers define the termini of the polynucleotide product of thepolymerization reaction; and wherein said means for thermally cyclingcomprises means for thermally cycling the contents of said chamberbetween a temperature controlled to dehybridize double strandedpolynucleotide thereby to produce single stranded polynucleotide, topermit annealing of said primers to complementary regions of singlestranded polynucleotide, and to permit synthesis of polynucleotidebetween said primers, thereby to amplify said preselectedpolynucleotide.
 3. The device of claim 1, wherein said solid substratecomprises microfabricated silicon.
 4. A device for amplifying apreselected polynucleotide in a sample, the device comprising: a solidsubstrate microfabricated to define; a sample inlet port; a flow systemfor micro- to picoliter volumes, comprising: a sample flow channelextending from said inlet port; and a polynucleotide amplificationreaction chamber, in fluid communication with said flow channel,containing a preselected polynucleotide and polynucleotide amplificationreagents; said chamber and said channel being of dissimilar dimension;and a fluid exit port in fluid communication with said flow system; andmeans for thermally cycling the contents of said chamber whereby, ineach cycle, temperature is controlled to dehybridize double strandedpolynucleotide, and to permit synthesis of polynucleotide, thereby toamplify said preselected polynucleotide.
 5. The device of claim 4,wherein said flow system further comprises a detection chamber in fluidcommunication with said amplification chamber.
 6. A method foramplifying a preselected polynucleotide in a sample by conducting apolynucleotide amplification reaction, the method comprising: (i)providing a device comprising: a solid substrate microfabricated todefine: a sample inlet port; a flow system for micro- to picolitervolumes comprising: a sample flow channel extending from said inletport; and a polynucleotide amplification reaction chamber in fluidcommunication with said flow channel; said chamber and said channelbeing of dissimilar dimension; and a fluid exit port in fluidcommunication with said flow system; and means for thermally regulatingthe contents of said chamber at a temperature controlled to permitamplification of said preselected polynucleotide; (ii) delivering,through said inlet port and said flow system to said reaction chamber, asample polynucleotide and reagents required for an in vitropolynucleotide amplification reaction; and (iii) thermally controllingthe contents of said chamber to permit amplification of saidpolynucleotide.
 7. The method of claim 6, wherein said amplificationreaction is a polymerase chain reaction (PCR); wherein in step (i), saidmeans for thermally controlling comprises means for thermally cyclingthe contents of said chamber; wherein step (ii) includes the step ofadding to said amplification chamber: a polymerase, nucleosidetriphosphates, a first primer hybridizable with said samplepolynucleotide, and a second primer hybridizable with a nucleic acidcomprising a sequence complementary to said polynucleotide, and whereinsaid first and second primers define the termini of the polynucleotideproduct of the polymerization reaction; and wherein step (iii) includesthe step of thermally cycling the contents of said chamber whereby, ineach cycle, the temperature is controlled to dehybridize double strandedpolynucleotide thereby to produce single stranded polynucleotide, topermit annealing of complementary regions of single strandedpolynucleotide, and to permit synthesis and polymerization ofpolynucleotide between said primers.
 8. A device for amplifying apreselected polynucleotide in a sample by conducting a polynucleotideamplification reaction, the device comprising: a solid substratemicrofabricated to define: a sample inlet port; a flow system for micro-to picoliter volumes, comprising: a sample flow channel extending fromsaid inlet port; and a polynucleotide amplification chamber, in fluidcommunication with said flow channel, said chamber containing reagentsfor amplifying a preselected polynucleotide in vitro; said chamber andsaid channel being of dissimilar dimension; and a fluid exit port influid communication with said flow system.
 9. The device of claim 8,wherein said reagents comprise reagents for conducting a polymerasechain reaction.
 10. A device for amplifying a preselected polynucleotidein a sample by conducting a polynucleotide amplification reaction, thedevice comprising: a solid substrate which is microfabricated to define:a sample inlet port; a flow system, comprising: a sample flow channelextending from said inlet port; and a polynucleotide amplificationreaction chamber in fluid communication with said flow channel; saidchamber and said channel being of dissimilar dimension; and a fluid exitport in fluid communication with said flow system; and means forthermally cycling the contents of said chamber whereby in each cycle thetemperature is controlled to dehybridize double stranded polynucleotideand to permit the in-vitro amplification of a preselectedpolynucleotide.
 11. The device of claim 10, wherein said amplificationreaction is a polymerase chain reaction (PCR), and wherein saidamplification chamber contains: a preselected polynucleotide, apolymerase, nucleoside triphosphates, a first primer hybridizable with asingle strand of said polynucleotide, and a second primer hybridizablewith a nucleic acid comprising a sequence complementary to said singlestrand, wherein said first and second primers define the termini of thepolynucleotide product of the polymerization reaction; and wherein saidmeans tar thermally cycling comprises means for thermally cycling thecontents of said chamber between a temperature controlled to dehybridizedouble stranded polynucleotide thereby to produce single strandedpolynucleotide, to permit annealing of said primers to complementaryregions of single stranded polynucleotide, and to permit synthesis ofpolynucleotide between said primers, thereby to amplify said preselectedpolynucleotide.
 12. The device of claim 10, wherein said solid substratecomprises microfabricated silicon.
 13. The device of claim 10, whereinsaid flow system further comprises a detection chamber in fluidcommunication with said amplification chamber.
 14. A device foramplifying a preselected polynucleotide in a sample, the devicecomprising: a solid substrate microfabricated to define: a sample inletport; a flow system for micro- to pico-liter volumes comprising: asample flow channel extending from said inlet port; and a polynucleotideamplification reaction chamber, in fluid communication with said flowchannel; said chamber and said channel being of dissimilar dimension;and a fluid exit port in fluid communication with said flow system; andmeans for thermally cycling the contents of said chamber whereby, ineach cycle, temperature is controlled to dehybridize double strandedpolynucleotide, and to permit synthesis of polynucleotide, thereby toamplify said preselected polynucleotide.
 15. A process for manufacturingon a wafer an instrument for controlling a chemical reaction, comprisingthe steps of etching said wafer to form a reaction chamber, anddepositing a resistive element on said wafer adjacent a boundary of saidreaction chamber.
 16. The process of claim 15 further comprising thesteps of etching a reagent chamber and etching a passage from saidreagent chamber to said reaction chamber.
 17. The process of claim 15further comprising the step of depositing a Lamb-wave transducer on saidwafer.
 18. The process of claim 17 wherein said Lamb-wave transducer islocated near t-boundary of said reaction chamber.
 19. The process ofclaim 15 further comprising the step of depositing a window of anelastomeric material interposed between a boundary of one of saidchambers and the exterior of said wafer.
 20. The process of claim 15wherein said wafer comprises a semiconductor.
 21. The process of claim20 wherein said semiconductor comprises a silicon-based material.